It is now thought that a major cause of infertility and subfertility in humans arises due to failure of implantation of the fertilised embryo within the uterus. Implantation failure also contributes to the difficulties encountered in in-vitro fertilisation programmes and in animal breeding.
Although hormonal manipulation is effective in certain selected cases, at present there is no uniformly successful therapy available to address this problem.
Australian Patent Application No 77189/87, relates to the discovery that the fertility of mammals can be controlled by causing artificial changes to the level (or activity) of the ether phospholipid Platelet Activating Factor (PAF) which is produced and secreted by mammalian embryos. Specifically the above application discloses that such changes can result in a contraceptive effect or fertility enhancement. The specification states that "to increase the chance of implantation it was necessary to increase the in vivo concentration of PAF, usually by the artificial addition of exogenous PAF or PAF analogue or by reducing the rate of destruction of PAF by acetylhydrolase" during the pre and peri-implantation period.
An increase in PAF concentration can be easily achieved in vitro simply by adding synthetic PAF. In vivo this is more difficult. Adding PAF to biological fluids results in its very rapid deactivation by serum acetylhydrolase and other systems. This fluid phase catabolism seems not to be of great significance for embryo-derived PAF since the evidence to date shows that it is bound, probably to a protein carrier, in such a manner as to make it not readily susceptible to the action of serum acetylhydrolase. The predominant route of embryo-derived PAF catabolism therefore is via cellular catabolism.
This route is thought to involve the binding of PAF to its plasma membrane receptor, and on cellular activation the PAF is internalised and cytosolic acetylhydrolase converts it to lyso-PAF followed soon after by the action of acyltransferase which leads to the production of a long chain acyl alkyl glycerophosphocholine which is not active biologically.
It is possible to overcome the lability of exogenously added PAF by the use of PAF analogues that have biological activity, but which are not susceptible to hydrolysis. A number of these have been reported but the one most fully studied to date is a carbamyl derivative. This compound is quite resistant to the actions of acetylhydrolase. Another alternative is to use embryo-derived PAF that has been generated in vitro and which is in a bound form and thus non-hydrolysable. This is a real possibility but the cost of production is likely to be prohibitive. Both of these approaches have the disadvantage that administration of exogenous PAF can be dangerous. It is a potent mediator of anaphylaxis and allergic responses. Therefore the systemic administration of PAF must be performed extremely carefully. Local administration of PAF into the uterus does not appear to expose the mother to these dangers (at least in the mouse and the sheep). Local administration of PAF however is of only limited therapeutic practicality, systemic administration is by far the most desirable.
The present invention comes from the entirely unexpected observation that a variety of pharmacological inhibitors of PAF activity have a bi-phasic effect on the establishment of pregnancy. At low dose (the dose where they are first effective at inhibiting the action of PAF) they inhibit implantation in both the mouse and rat. This effect reaches a maximum and further increases, (by as little as 4-10 fold concentration) result in a loss of this contragestational action and enhancement of implantation. This is entirely unexpected because for all other PAF mediated patho-physiological events investigated to date, the actions of the pharmacological inhibitors of PAF are active at even massive doses. In no cases in the literature of which the inventor is aware is there evidence for such apparent partial agonism. Recent data has shown that PAF receptor antagonists act to inhibit the cellular catabolism of PAF. This has been demonstrated to occur for at least the platelet, neutrophil and endothelium.
To date this effect has only been shown to occur for PAF receptor antagonists however and it has been proposed that the action is to prevent binding of PAF to its receptor and hence limit the amount of PAF that is internalised. The present invention shows that this phenomenon is not limited to receptor antagonists, in fact, a number of classes of pharmacological inhibitors of PAF also inhibit PAF catabolism. This inhibitory activity for all classes of compounds investigated occurred at concentrations somewhat higher than that which inhibited the cell activating effects of PAF. Thus, the pharmacological action would appear to be that at a low dose the agents inhibit PAF activation of cells but at a higher dose they also limit the catabolism of PAF. The result would therefore be a tendency for the build up of PAF. In most patho-physiological cases the PAF would be susceptible to fluid phase catabolism and therefore there is little net effect on the antagonistic action of these agents. In the case of embryo-derived PAF however the PAF is in a form that is not readily catabolised in the fluid phase and therefore a localised build up in the concentration of PAF would result. The inventor has now shown that the primary site of action of PAF on embryos is on the embryo itself in a positive autocrine manner, this localised build up results in PAF out-competing the inhibitors effects on the embryo and causing enhancement of fertility.
Apart from the reduction in catabolism of PAF, and the consequent increase in its local concentration, there is an associated benefit. The high concentration of antagonists in the body means that the systemic actions of PAF are suppressed. Therefore, PAF can be administered exogenously without the normal deleterious side effects. This systemic administration helps in elevating the local concentration of PAF in the reproductive tract and hence fertility.
Thus the present invention relates specifically to fertility enhancement, and in one aspect provides a novel way of overcoming the practical problems of the prior art involved in the use of PAF or analogues thereof for the enhancement of fertility.